中华绒螯蟹精子膜蛋白的提取分离及部分生化性质研究
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摘要
成熟精子的质膜表面含有多种蛋白，有些是精子本身所固有的，有些是在精子成熟过程中黏附到精子表面的，这些精子膜蛋白在维持精子形态结构完整、完成精子的新陈代谢和生殖功能中发挥着重要的作用，特别是与精卵识别、结合以及质膜融合等受精活动密切相关。
本文以我国重要的淡水养殖珍品——中华绒螯蟹(Eriocheir sinensis)为实验材料，从提取、分离、生化性质分析等几个方面对精子膜蛋白进行了初步的研究。首先，通过分级分离和比较电泳的方法对中华绒螯蟹精荚各部分的蛋白组分进行分析，并在试验过程中，建立起一套精荚分级分离的方法，通过电泳分析和透射电镜观察等手段对这一方法的可行性进行了验证。其次，利用超声波匀浆和蔗糖密度梯度离心的方法对中华绒螯蟹的精子膜进行了分离纯化，并利用透射电镜技术对精子膜分离纯化的效果进行了研究。最后，利用去污剂溶脱的方法从中华绒螯蟹精子膜表面提取得到膜蛋白，并对其部分生化性质进行研究。
实验结果显示，通过分级分离所得的精液、精荚基质和纯品精子等各个部分，在SDS-PAGE电泳图谱上均显示出不同的特征性谱带，且试验的重复性较好，证明本实验建立的精荚分级分离的方法是可行的。对精液、精荚基质蛋白组分的PAS糖蛋白染色结果显示，精液中主要的蛋白组分均为糖蛋白，共有3种；精荚基质中有2种主要的蛋白组分是糖蛋白。这一实验结果与精液的营养功能一致，同时也表明，精荚基质除了对精子具有保护作用外，还可能含有精子在其中进行新陈代谢所需要的营养物质。通过对精荚和分级分离得到的精子样品进行透射电镜观察，研究发现，分级分离所得的精子绝大多数形态结构未发生变化，质膜结构保持完整，这一结果进一步证明了通过分级分离方法获取精子的可行性，也为接下来的膜蛋白提取研究奠定了基础。另外，利用超声波匀浆和蔗糖密度梯度离心法成功地纯化得到了中华绒螯蟹的精子膜，经透射电镜观察发现，所得的精子膜均一性很好。使用含1％ TritonX-100和少量SDS的膜蛋白提取缓冲液对中华绒螯蟹精子膜蛋白进行溶脱提取，对所得的膜蛋白溶液进行SDS-PAGE电泳分析，结果显示，中华绒螯蟹的精子至少含有11种不同的膜多肽组分。对这些精子膜
摘要
蛋白组分的部分生化性质进行分析，结果表明，中华绒鳌蟹精子膜蛋白是一组具
酸性等电点的低分子量糖蛋白，分子量在38ku一83ku之间，等电点在4.8左右。
本文在中华绒鳌蟹精子膜的分离纯化，精子膜蛋白的提取分离，及其生化性
质的测定分析等方面进行了初步的研究和探索，各种精子膜蛋白在受精过程中的
具体作用，仍有待于进一步的研究。
Mature sperms have many kinds of proteins in it's plasma membrane. Some of them are sperm-specific, others bind to the sperm surface during the period of sperm maturation. These sperm membrane proteins(SMPs) play important role in the maintenance of sperm's shape and structure, the metabolism of sperms and the reproduction, especially in the fertilization.
With Eriocheir sinensis as the experiment material, SMPs were studied in such respects as extraction, isolation and biochemical characteristics analysis. Firstly, the Protein compositions of various parts of the spermatophore were studied through the methods of fractionation and comparing PAGE. And a method for spermatophore fractionation was established, the feasibility of this method was examined by the means of PAGE and transmission electronic microscopy(TEM). Then, the sperm membrane was purified with the method of sucrose density gradients centrifugation, and the effect on it by isolation was observed in the transmission electronic microscope. Finally, SMPs were extracted from the surface of E. sinensis sperms by the use of decontaminant, and characterization of SMPs from E. sinensis were conducted.
The result of SDS-PAGE showed that different parts in spermatophore, including spermatophore matrix, seminal plasma and the whole sperm, had different characteristic bands in PAGE maps, and the result could be repeated, so the method for spermatophore fractionation was reliable. In the Staining PAGE map of glycoproteins from seminal plasma and spermatophore matrix, there were 3 kinds of glycoproteins in seminal plasma, and 2 in spermatophore matrix. So it could be inferred that seminal plasma and spermatophore matrix had nutritional effect on
sperms. But it would need further study to clarify the real function of spermatophore matrix. Under TEM ,most of the sperms kept intact after fractionation ,which proved the feasibility of the method on microstructure level and made the basis of the following experiment. Furthermore, with the method of ultrasonic wave homogenization and sucrose density gradients centrifugation, sperm membrane of E. sinensis was purified with good homogeneity successfully.
Extraction buffers with 1% TritonX-100 and low concentration of SDS was applied to dissolve membrane proteins off E. sinensis's sperms. The membrane Protein composition was analyzed by SDS-PAGE. As SDS-PAGE maps showed , there were at least 11 kinds of SMPs in E. sinensis, the composition of SMPs is obviously different from that of the demembranated sperms. Some biochemical characteristics of SMPs in E. sinensis were studied in this paper: SMPs in E. sinensis were a group of glycoproteins, molecular weights of which between 38ku and 83ku, isoelectric points (PI) of which are all about 4.8.
本文以我国重要的淡水养殖珍品——中华绒螯蟹(Eriocheir sinensis)为实验材料，从提取、分离、生化性质分析等几个方面对精子膜蛋白进行了初步的研究。首先，通过分级分离和比较电泳的方法对中华绒螯蟹精荚各部分的蛋白组分进行分析，并在试验过程中，建立起一套精荚分级分离的方法，通过电泳分析和透射电镜观察等手段对这一方法的可行性进行了验证。其次，利用超声波匀浆和蔗糖密度梯度离心的方法对中华绒螯蟹的精子膜进行了分离纯化，并利用透射电镜技术对精子膜分离纯化的效果进行了研究。最后，利用去污剂溶脱的方法从中华绒螯蟹精子膜表面提取得到膜蛋白，并对其部分生化性质进行研究。
实验结果显示，通过分级分离所得的精液、精荚基质和纯品精子等各个部分，在SDS-PAGE电泳图谱上均显示出不同的特征性谱带，且试验的重复性较好，证明本实验建立的精荚分级分离的方法是可行的。对精液、精荚基质蛋白组分的PAS糖蛋白染色结果显示，精液中主要的蛋白组分均为糖蛋白，共有3种；精荚基质中有2种主要的蛋白组分是糖蛋白。这一实验结果与精液的营养功能一致，同时也表明，精荚基质除了对精子具有保护作用外，还可能含有精子在其中进行新陈代谢所需要的营养物质。通过对精荚和分级分离得到的精子样品进行透射电镜观察，研究发现，分级分离所得的精子绝大多数形态结构未发生变化，质膜结构保持完整，这一结果进一步证明了通过分级分离方法获取精子的可行性，也为接下来的膜蛋白提取研究奠定了基础。另外，利用超声波匀浆和蔗糖密度梯度离心法成功地纯化得到了中华绒螯蟹的精子膜，经透射电镜观察发现，所得的精子膜均一性很好。使用含1％ TritonX-100和少量SDS的膜蛋白提取缓冲液对中华绒螯蟹精子膜蛋白进行溶脱提取，对所得的膜蛋白溶液进行SDS-PAGE电泳分析，结果显示，中华绒螯蟹的精子至少含有11种不同的膜多肽组分。对这些精子膜
摘要
蛋白组分的部分生化性质进行分析，结果表明，中华绒鳌蟹精子膜蛋白是一组具
酸性等电点的低分子量糖蛋白，分子量在38ku一83ku之间，等电点在4.8左右。
本文在中华绒鳌蟹精子膜的分离纯化，精子膜蛋白的提取分离，及其生化性
质的测定分析等方面进行了初步的研究和探索，各种精子膜蛋白在受精过程中的
具体作用，仍有待于进一步的研究。
Mature sperms have many kinds of proteins in it's plasma membrane. Some of them are sperm-specific, others bind to the sperm surface during the period of sperm maturation. These sperm membrane proteins(SMPs) play important role in the maintenance of sperm's shape and structure, the metabolism of sperms and the reproduction, especially in the fertilization.
With Eriocheir sinensis as the experiment material, SMPs were studied in such respects as extraction, isolation and biochemical characteristics analysis. Firstly, the Protein compositions of various parts of the spermatophore were studied through the methods of fractionation and comparing PAGE. And a method for spermatophore fractionation was established, the feasibility of this method was examined by the means of PAGE and transmission electronic microscopy(TEM). Then, the sperm membrane was purified with the method of sucrose density gradients centrifugation, and the effect on it by isolation was observed in the transmission electronic microscope. Finally, SMPs were extracted from the surface of E. sinensis sperms by the use of decontaminant, and characterization of SMPs from E. sinensis were conducted.
The result of SDS-PAGE showed that different parts in spermatophore, including spermatophore matrix, seminal plasma and the whole sperm, had different characteristic bands in PAGE maps, and the result could be repeated, so the method for spermatophore fractionation was reliable. In the Staining PAGE map of glycoproteins from seminal plasma and spermatophore matrix, there were 3 kinds of glycoproteins in seminal plasma, and 2 in spermatophore matrix. So it could be inferred that seminal plasma and spermatophore matrix had nutritional effect on
sperms. But it would need further study to clarify the real function of spermatophore matrix. Under TEM ,most of the sperms kept intact after fractionation ,which proved the feasibility of the method on microstructure level and made the basis of the following experiment. Furthermore, with the method of ultrasonic wave homogenization and sucrose density gradients centrifugation, sperm membrane of E. sinensis was purified with good homogeneity successfully.
Extraction buffers with 1% TritonX-100 and low concentration of SDS was applied to dissolve membrane proteins off E. sinensis's sperms. The membrane Protein composition was analyzed by SDS-PAGE. As SDS-PAGE maps showed , there were at least 11 kinds of SMPs in E. sinensis, the composition of SMPs is obviously different from that of the demembranated sperms. Some biochemical characteristics of SMPs in E. sinensis were studied in this paper: SMPs in E. sinensis were a group of glycoproteins, molecular weights of which between 38ku and 83ku, isoelectric points (PI) of which are all about 4.8.
引文
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